Sequential Solid State Research Articles

Coproduction of Microbial Oil and Carotenoids within the Circular Bioeconomy Concept: A Sequential Solid-State and Submerged Fermentation Approach

The main objective of integrative biorefinery platforms is to propose efficient green methodologies addressed to obtain high-value compounds with low emissions through biochemical conversions. This work first screened the capacity of various oleaginous yeast to cosynthesize high-value biomolecules such as lipids and carotenoids. Selected strains were evaluated for their ability to coproduce such biocompounds in the waste-based media of agro-food (brewer’s spent grain, pasta processing waste and bakery waste). Carbon and nitrogen source feedstock was obtained through enzymatic hydrolysis of the agro-food waste, where up to 80% of total sugar/starch conversion was obtained. Then, the profitability of the bioprocess for microbial oil (MO) and carotenoids production by Sporobolomyces roseus CFGU-S005 was estimated via simulation using SuperPro Designer®. Results showed the benefits of establishing optimum equipment scheduling by identifying bottlenecks to increase profitability. Sensitivity analysis demonstrated the impact of MO price and batch throughput on process economics. A profitable process was achieved with a MO batch throughput of 3.7 kg/batch (ROI 31%, payback time 3.13 years). The results revealed areas that require further improvement to achieve a sustainable and competitive process for the microbial production of carotenoids and lipids.

Screening of Lignocellulolytic Enzyme Activities in Fungal Species and Sequential Solid-State and Submerged Cultivation for the Production of Enzyme Cocktails.

Various fungal species can degrade lignocellulolytic materials with their enzyme cocktails composed of cellulolytic and lignolytic enzymes. In this work, seven fungal species (Mucor indicus DSM 2185, Paecilomyces variotii CBS 372.70, Myceliophthora thermophila CBS 663.74, Thielavia terrestris CBS 456.75, Botryosphaeria dothidea JCM 2738, Fusarium oxysporum f.sp. langenariae JCM 9293, and Fusarium verticillioides JCM 23107) and four nutrient media were used in the screening for effective lignocellulose degrading enzymes. From the seven tested fungi, F. oxysporum and F. verticilliodes, along with nutrient medium 4, were selected as the best medium and producers of lignocellulolytic enzymes based on the determined xylanase (>4 U mg−1) and glucanase activity (≈2 U mg−1). Nutrient medium 4 supplemented with pretreated corn cobs was used in the production of lignocellulolytic enzymes by sequential solid-state and submerged cultivation of F. oxysporum, F. verticilliodes, and a mixed culture of both strains. F. oxysporum showed 6 times higher exoglucanase activity (3.33 U mg−1) after 5 days of cultivation in comparison with F. verticillioides (0.55 U mg−1). F. oxysporum also showed 2 times more endoglucanase activity (0.33 U mg−1). The mixed culture cultivation showed similar endo- and exoglucanase activities compared to F. oxysporum (0.35 U mg−1; 7.84 U mg−1). Maximum xylanase activity was achieved after 7 days of cultivation of F. verticilliodes (≈16 U mg−1), while F. oxysporum showed maximum activity after 9 days that was around 2 times lower compared to that of F. verticilliodes. The mixed culture achieved maximum xylanase activity after only 4 days, but the specific activity was similar to activities observed for F. oxysporum. It can be concluded that both fungal strains can be used as producers of enzyme cocktails for the degradation of lignocellulose containing raw materials, and that corn cobs can be used as an inducer for enzyme production.

Potential Role of Sequential Solid-State and Submerged-Liquid Fermentations in a Circular Bioeconomy

An efficient processing of organic solid residues will be pivotal in the development of the circular bioeconomy. Due to their composition, such residues comprise a great biochemical conversion potential through fermentations. Generally, the carbohydrates and proteins present in the organic wastes cannot be directly metabolized by microorganisms. Thus, before fermentation, enzymes are used in a hydrolysis step to release digestible sugars and nitrogen. Although enzymes can be efficiently produced from organic solid residues in solid-state fermentations (SsF), challenges in the development and scale-up of SsF technologies, especially bioreactors, have hindered a wider application of such systems. Therefore, most of the commercial enzymes are produced in submerged-liquid fermentations (SmF) from expensive simple sugars. Instead of independently evaluating SsF and SmF, the review covers the option of combining them in a sequential process in which, enzymes are firstly produced in SsF and then used for hydrolysis, yielding a suitable medium for SmF. The article reviews experimental work that has demonstrated the feasibility of the process and underlines the benefits that such combination has. Finally, a discussion is included which highlights that, unlike typically perceived, SsF should not be considered a counterpart of SmF but, in contrast, the main advantages of each type of fermentation are accentuated in a synergistic sequential SsF-SmF.

Protein improvement of banana peel through sequential solid state fermentation using mixed-culture of Phanerochaete chrysosporium and Candida utilis.

Banana peel (BP) is a major waste produced by fruit processing industries. Pre-treatment of BP at different temperatures led to 40% reduction in saponin at 100°C (from 9.5 to 5.7mg/g). Sequential mixed culture of Phanerochaete chrysosporium (P. chrysosporium) and Candida utilis (C. utilis) gave highest protein enrichment (88.93mg/g). There is 26% increase in protein synthesis (from 88.93 to 111.78mg/g) after media screening. Inclusion of KH2PO4, FeSO4·7H2O, wheat flour and sucrose in the media contributed positively to protein synthesis, while elevated concentration of urea, peptone, K2HPO4, KCl, NH4H2PO4, and MgSO4.7H2O are required to reach optimum protein synthesis. Total soluble sugar (TSS), total reducing sugar (TRS) and total carbohydrate (CHO) consumption varied with respect to protein synthesis in all experimental runs. Optimum protein synthesis required 6 days and inclusion of 5% sucrose, 0.6% NH4H2PO4, 0.4% KCl, and 0.5% MgSO4·7H2O as concentration media constituents to reach 140.95mg/g protein synthesis equivalent to 300% increase over the raw banana peel protein content (35.0mg/g).

Selective conversion of cassava mash to glucose using solid acid catalysts by sequential solid state mixed-milling reaction and thermo-hydrolysis

Solid acid catalysts have been recently studied in starch thermo-hydrolysis owing to their high catalytic activity, recyclability, ease to separate and environmental advantages. However, the solid state reaction of effective catalyst and starch molecules requires specific interaction between active sites in catalyst and α,1-4 glycosidic bonds in starch. Therefore, in this study catalyst mixed-milling was conducted prior to thermal hydrolysis to enhance glucose production efficiency. The results showed that catalyst mixed-milling process (24 h) followed by thermo-hydrolysis at 140 °C for 6 h using HA-L-SO3H gave highest starch conversion of 93.72% corresponding to 37.45% glucose yield and 83% selectivity. From the kinetic study, the rate constant of cassava mash-to-oligomers conversion (k1) using catalyst mixed-milling was 1.77 times higher than ball-milling without adding catalyst. This indicated that mixed-milling solid state reaction with effective catalyst significantly provided complete cassava mash conversion and enhanced selectivity as well as rate of starch depolymerization reaction.

On-Site Production of Cellulolytic Enzymes by the Sequential Cultivation Method.

The conversion of renewable lignocellulosic biomass into fuels, chemicals, and high-value materials using the biochemical platform has been considered the most sustainable alternative for the implementation of future biorefineries. However, the high cost of the cellulolytic enzymatic cocktails used in the saccharification step significantly affects the economics of industrial large-scale conversion processes. The on-site production of enzymes, integrated to the biorefinery plant, is being considered as a potential strategy that could be used to reduce costs. In such approach, the microbial production of enzymes can be carried out using the same lignocellulosic biomass as feedstock for fungal development and biofuels production. Most of the microbial cultivation processes for the production of industrial enzymes have been developed using the conventional submerged fermentation. Recently, a sequential solid-state followed by submerged fermentation has been described as a potential alternative cultivation method for cellulolytic enzymes production. This chapter presents the detailed procedure of the sequential cultivation method, which could be employed for the on-site production of the cellulolytic enzymes required to convert lignocellulosic biomass into simple sugars.

Direct Succinic Acid Production from Minimally Pretreated Biomass Using Sequential Solid-State and Slurry Fermentation with Mixed Fungal Cultures

Conventional bio-based succinic acid production involves anaerobic bacterial fermentation of pure sugars. This study explored a new route for directly producing succinic acid from minimally-pretreated lignocellulosic biomass via a consolidated bioprocessing technology employing a mixed lignocellulolytic and acidogenic fungal co-culture. The process involved a solid-state pre-fermentation stage followed by a two-phase slurry fermentation stage. During the solid-state pre-fermentation stage, Aspergillus niger and Trichoderma reesei were co-cultured in a nitrogen-rich substrate (e.g., soybean hull) to induce cellulolytic enzyme activity. The ligninolytic fungus Phanerochaete chrysosporium was grown separately on carbon-rich birch wood chips to induce ligninolytic enzymes, rendering the biomass more susceptible to cellulase attack. The solid-state pre-cultures were then combined in a slurry fermentation culture to achieve simultaneous enzymatic cellulolysis and succinic acid production. This approach generated succinic acid at maximum titers of 32.43 g/L after 72 h of batch slurry fermentation (~10 g/L production), and 61.12 g/L after 36 h of addition of fresh birch wood chips at the onset of the slurry fermentation stage (~26 g/L production). Based on this result, this approach is a promising alternative to current bacterial succinic acid production due to its minimal substrate pretreatment requirements, which could reduce production costs.

Effect of a novel method for in-house cellulase production on 2G ethanol yields

Abstract Significant enzyme loadings are needed in the production of second-generation (2G) ethanol from biomass, and the cost of these enzymes remains a critical challenge for implementation of industrial plants at commercial scale. Aiming to contribute to 2G ethanol viability, its production yield was investigated in terms of the fermentability of sugarcane bagasse hydrolysates obtained using enzymatic cocktails produced in-house. For that, two different cultivation methods (sequential solid-state and submerged fermentation and conventional submerged fermentation) were compared. Sequential and submerged fermentation methods resulted in enzymatic cocktails with different compositions in terms of endoglucanase, xylanase, and β-glucosidase activities. At the same protein loading, use of the sequential fermentation cocktail resulted in 11% hydrolysis increase, relative to that achieved with the submerged fermentation cocktail. Sugarcane bagasse hydrolysis was increased by 8–19% with the addition of low loadings of in-house produced cocktails to a commercial enzyme. Furthermore, the yield of 2G ethanol production (78%) from the hydrolysate obtained using the sequential fermentation cocktail was higher than the yields achieved using the conventional submerged fermentation cocktail hydrolysate (71%) and the commercial enzyme hydrolysate (74%). The sequential fermentation method was superior to conventional submerged fermentation, resulting in a cocktail with higher enzyme activities and better performance in sugarcane bagasse hydrolysis. Moreover, this work shows the improvement on 2G ethanol production by the sequential fermentation process developed for in-house enzyme production. These findings contribute to existing knowledge of 2G ethanol production by demonstrating the effect of the enzymatic cocktails composition on the fermentability of sugarcane bagasse hydrolysates.

Sequential Solid-State and Submerged Cultivation of the White Rot Fungus Pleurotus ostreatus on Biomass and the Activity of Lignocellulolytic Enzymes

Sequential solid-state and submerged cultivation with various lignocellulosic biomasses as a substrate for lignocellulolytic enzyme production by Pleurotus ostreatus were assessed by measuring endoglucanase, xylanase, and laccase activities. An unconventional pre-culture method was established by cultivating the P. ostreatus mycelia in a solid substrate medium for an initial fungal growth phase, followed by a transition to submerged fermentation through adding a liquid culture medium. The lignocellulolytic enzymes of P. ostreatus in different fermentation methods revealed wide differences. The higher yields of endoglucanase (3152 ± 139 U/L), xylanase (3064 ± 40 U/L), and laccase (543 ± 21 U/L) were achieved by using the sequential solid-state submerged method compared to conventional solid-state and submerged cultivation. Generally speaking, sequential solid-state and submerged fermentation of cottonseed hull is favorable for laccase secretion, whereas sequential solid-state and submerged fermentation of corncob provides better production of hydrolytic enzymes. These results revealed that the nature of the lignocellulosic biomass and the fermentation method play an important role in the expression of lignocellulolytic enzymes. This indication would be helpful in optimizing the production of integrated industrial lignocellulolytic enzymes.

Validation of a novel sequential cultivation method for the production of enzymatic cocktails from Trichoderma strains.

The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry.

Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulase

Sequential solid-state and submerged cultivation with sugarcane bagasse as substrate for cellulase production by Aspergillus niger A12 was assessed by measuring endoglucanase activity. An unconventional pre-culture with an initial fungal growth phase under solid-state cultivation was followed by a transition to submerged fermentation by adding the liquid culture medium to the mycelium grown on solid substrate. For comparison, control experiments were conducted using conventional submerged cultivation. The cultures were carried out in shake flasks and in a 5-L bubble column bioreactor. An endoglucanase productivity of 57±13IU/L/h was achieved in bubble column cultivations prepared using the new method, representing an approximately 3-fold improvement compared to conventional submerged fermentation. Therefore, the methodology proposed here of a sequential fermentation process offers a promising alternative for cellulase production.

Conversion of cassava flour to fuel ethanol by sequential solid state and submerged cultures

Abstract A process for conversion of cassava flour to ethanol was developed. This involved direct inoculation of Aspergillus awamori spores into a cassava flour paste and incubation for some period during which hydrolytic enzymes are produced (solid state culture or koji production) and subsequent addition of water and yeast cells, during which there is simultaneous hydrolysis and ethanol production (submerged culture). When cassava flour alone was used for the solid state phase, the paste was very sticky, making mixing and aeration difficult. However, addition of rice bran improved the texture and enzyme production. The optima rice bran concentration, spore inoculum concentration, and duration of solid state culture before submerged culture were 20%, 6.16 × 10 6 spores/100 g, and 2 days, respectively. Under these optimum conditions, a high ethanol concentration of 120 g/L and ethanol yield of 0.309 g-ethanol/g-cassava flour were obtained. This ethanol yield corresponds to 0.44 g-ethanol/g-cassava starch.

In-Situ Annealing Transmission Electron Microscopy Study of Pd/Ge/Pd/GaAs Interfacial Reactions

AbstractIn-situ TEM annealing experiments on the Pd (20 nm) / a-Ge (150 nm) / Pd (50 nm) GaAs ohmic contact system have permitted real time determination of the evolution of contact microstructure. As-deposited cross-sectional samples of equal thickness were prepared using a focused ion beam (FIB) method and then subjected to in-situ annealing at temperatures between 130-400 °C. Excluding Pd-GaAs interactions, four sequential solid state reactions were observed during annealing of the Pd:Ge thin films. First, interdiffusion of the Pd and Ge layers occurred, followed by formation of the hexagonal Pd2Ge phase. This hexagonal phase then transformed into orthorhombic PdGe, followed by solid state epitaxial growth of Ge at the contact / GaAs interface. The kinetics of the solid state reactions, which occur during ohmic contact formation, were determined by measuring the grain growth rates associated with each phase from the videotape observations. These data agreed with a previous study that measured the activation energies through a differential scanning calorimetry (DSC) method. We established that the Ge transport to the GaAs interface was dependent upon the grain size of the PdGe phase. The nucleation and growth of this phase was demonstrated to have a significant effect on the solid phase epitaxial growth of Ge on GaAs. These findings allowed us to engineer an improved two step annealing procedure that would control the shape and size of the PdGe grains. Based on these results, we have established the suitability of combining FIB sample preparation with in-situ cross-sectional transmission electron microscopy (TEM) annealing for studying thin film solid-state reactions.

A reciprocating canister superconducting magnetic separator

This paper describes a small scale Reciprocating Canister Superconducting Magnetic Separator (RCSMS) that has been used to reduce the iron and potassium levels from a slurry of coarse English Kaolin. The magnet was a 5T solenoidal laboratory superconducting magnet, with a 0.127m warm bore, cooled by a BOC TurBOCool helium liquifier operating as a closed circuit refrigerator. The RCSMS was fully automated, controlled by a sequential solid state controller, and operated successfully for the duration of the experimental program.

A Precision Sequential Solid State Control Unit-An Electronic Control for Resistance Welding by Digital Technique

An electronic digital solid state control unit for a spot-welder has been developed to control the durations of its four operations, viz. ‘squeeze’, ‘weld’, ‘forge’ and ‘off’, precisely and in synchronism with the mains frequency. Periods of each of these operations can be set as desired, in steps of 1-cycle duration, anywhere from 1 to 100 cycles duration of the supply mains frequency. Furthermore, during the chosen weld period, a better control of the weld heat can be obtained by setting the firing angle of ignitrons. Provision has been made for automatic repetitive or non-repetitive welding.This unit uses all components indigenously manufactured except for two SCRs.This unit can be easily adapted for application in any other process which requires number of sequential operations involving precise control of duration of each operation.